Samples of peripheral whole blood are easily obtained from any of a wide variety of organisms, and thus blood would seem to be a rich source of material for gene expression studies. However, the composition of blood presents unusual challenges to the detection of nucleic acids from whole blood samples.
For example, whole blood includes a number of different cell types, including red blood cells (erythrocytes), platelets, and white blood cells (leukocytes). The white blood cells themselves include a variety of cell types, for example, granulocytes, such as neutrophils, basophils, and eosinophils, and mononuclear cells, such as monocytes and lymphocytes (including, e.g., T lymphocytes, B lymphocytes, and natural killer cells). Furthermore, when considering gene expression, only particular forms of particular white blood cell types may be of interest: e.g., active granular natural killer cells, Th-lymphocytes, or activated neutrophils, eosinophils, or basophils, to name only a few of the possible examples. Considering that red blood cells are estimated to occupy about 40-45% of the total blood volume while white blood cells and platelets together occupy only about 1-2% of the total blood volume, the difficulty of detecting a nucleic acid that is expressed only in white blood cells, or only in a particular subset or type of white blood cells, becomes clear.
Detection of nucleic acids from whole blood is further complicated, for example, by the high concentration of protein in blood (e.g., of hemoglobin from the red blood cells and of plasma proteins such as albumin, fibrinogen, and globulins) and by the prevalence of certain nucleic acids, particularly globin mRNA.
Current methods for analysis of gene expression in blood involve isolation of a particular type or group of cells (e.g., by red blood cell lysis, or by centrifugation to obtain peripheral blood mononuclear cells (PBMC)), purification of RNA from blood cells, and/or enzymatic manipulation (e.g., reverse transcription and/or target amplification) of the nucleic acids to be detected.
Among other aspects, the present invention provides methods for nucleic acid detection from whole blood that overcome the above noted difficulties. A complete understanding of the invention will be obtained upon review of the following.